Data Sheet 1_Rapid detection of Mycoplasma pneumoniae CARDS toxin in clinical respiratory specimens by a loop-mediated isothermal amplification assay.docx

In light of the absence of rapid and precise diagnostic laboratory tests for the detection of Mycoplasma pneumoniae (MP), a prominent etiological agent implicated in a range of respiratory infections, we developed and evaluated a rapid and straightforward loop-mediated isothermal amplification (LAMP) assay targeting the MP community-acquired respiratory distress syndrome toxin (CARDS TX) gene. The LAMP assay was performed at 65°C for a duration of 60 min, yielding a minimum detection concentration of MP CARDS TX at 0.4986 pg/μl. The assay exhibited no cross-reactivity with 13 other prevalent pathogens associated with respiratory infections or with other common bacterial toxin genes. To further substantiate the validity of the LAMP assay, 200 pharyngeal swabs or bronchoalveolar lavage (BAL) samples were collected from inpatients diagnosed with community-acquired pneumonia (CAP) between June 2021 and July 2022. The results were compared with those obtained by the quantitative real-time polymerase chain reaction (qPCR) method for verification purposes. Of the 200 clinical specimens, 11 exhibited positive results for MP by LAMP and 10 displayed positive results for MP by qPCR (P = 1.000). In summary, a sensitive, specific, straightforward, and expeditious LAMP method for CARDS TX identification was developed to facilitate rapid detection of MP in point-of-care settings. This assay enables early and accurate diagnosis, even in resource-limited environments, which is important for proper antibiotic treatment and prognosis of MP infection.

鉴于目前缺乏用于检测肺炎支原体（Mycoplasma pneumoniae, MP）的快速精准实验室诊断方法——肺炎支原体是引发多种呼吸道感染的重要致病原，我们开发并评估了一种靶向肺炎支原体社区获得性呼吸窘迫综合征毒素（CARDS TX）基因的快速简便环介导等温扩增（loop-mediated isothermal amplification, LAMP）检测方法。该LAMP检测方法在65℃条件下反应60分钟，对MP CARDS TX的最低检测浓度可达0.4986 pg/μl。该检测方法与13种其他常见呼吸道感染相关病原体及其他常见细菌毒素基因均无交叉反应。为进一步验证该LAMP检测方法的有效性，我们于2021年6月至2022年7月间，从确诊为社区获得性肺炎（community-acquired pneumonia, CAP）的住院患者中采集了200份咽拭子或支气管肺泡灌洗液（bronchoalveolar lavage, BAL）样本。将该方法的检测结果与定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）的检测结果进行比对验证。在200份临床样本中，LAMP法检出11份MP阳性样本，qPCR法检出10份MP阳性样本（P=1.000）。综上，本研究开发了一种灵敏、特异、简便且快速的CARDS TX基因检测LAMP方法，可用于临床即时检测场景下的MP快速检测。该方法即便在资源受限的环境中也可实现早期精准诊断，这对于MP感染的合理抗菌治疗及预后判断具有重要意义。